The overall goal of this proposal is to gain a better understanding of the function of Ras-related Ral proteins. RalA and RalB are present in the membranes of cytoplasmic vesicles including those involved in endocytosis and exocytosis, although a significant proportion of the proteins is also associated with the plasma membrane. As members of the Ras superfamily of GTPases, Ral proteins cycle between the active GTP bound and inactive GDP bound states. They are activated in cells upon binding to unique nucleotide exchange factors, Ral-GDS and RGL, and are inactivated by binding to a unique GTPase activating protein, Ral-GAP. We have recently shown that Ras can activate Ral in cells by binding to and activating its exchange factor Ral-GDS. Thus activation of Ral-GDS and Ral represents a newly identified downstream signalling pathway from Ras. Studying Ral function may aid in our understanding of how Ras proteins influence cell proliferation, differentiation and expression of differentiated cell function. We have also recently identified two downstream targets that interact with distinct regions of Ral proteins. These include a phospholipase D (PLD) that is stimulated by the Ras signalling pathway and Ral binding protein 1 (RalBP1), a novel GAP for Cdc42 and Rac GTPases. This proposal focuses on the molecular details of how Ral is regulated by Ras and possibly other upstream signals. It also investigates the function of PLD and RalBP1, and how these two signalling molecules are regulated by Ral. Finally, the experiments described in this proposal will determine whether one of the important functions of this newly identified Ras/Ral signalling pathway is to modulate cellular secretion and/or endocytosis.